Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins

Membrane proteins play key roles in most crucial cellular processes, ranging from cell-to-cell communication to signaling processes. Despite recent improvements, the expression of functionally folded membrane proteins in sufficient amounts for functional and structural characterization remains a challenge. Indeed, it is still difficult to predict whether a protein can be overproduced in a functional state in some expression system(s), though studies of high-throughput screens have been published in recent years. Prokaryotic expression systems present several advantages over eukaryotic ones. Among them, Lactococcus lactis (L. lactis) has emerged in the last two decades as a good alternative expression system to E. coli. The purpose of this chapter is to describe L. lactis and its tightly inducible system, NICE, for the effective expression of membrane proteins from both prokaryotic and eukaryotic origins.


Introduction
Membrane proteins (MPs), key proteins in cell physiology and drug targets, are encoded by one-third of the human genome [1,2]. MPs have different features: (i) they form various topologies, from peripheral to intrinsic polytopic proteins with a high number of transmembrane helices, (ii) their surface is relatively hydrophobic, (iii) detergents are required for their solubilization from the cell membrane, and they often need to be reconstituted into proteoliposomes for functional studies, (iv) they are flexible and unstable, (v) they must be targeted to the membrane for proper folding, (vi) they are expressed at very low levels, and/or (vii) they are functional in homo-and hetero-oligomeric states [3,4]. In order to increase and deepen our knowledge, in particular for pharmaceutical purposes, there is an increasing need for structural and functional studies [5]. During the last 7 years, the number of unique 3D structures of MPs increased from 400 to 1348 (https://blanco.biomol.uci.edu/mpstruc/, accessed on 21 December 2021), which is still far away from the 75,000 structures available for soluble proteins. The reason why the number of 3D structures is still so low is linked to the difficulty of obtaining sufficient amounts of functionally folded MPs. Functional and structural studies require high amounts of proteins. Therefore, the low concentration of MPs in cells highlights the need for heterologous expression systems. There are different types of expression systems such as cell-free systems [6], prokaryotic systems (E. coli and L. lactis), and eukaryotic expression systems (yeasts, plants, mammalian, or insect cells). All of them have advantages and drawbacks [3,4,7]. Bacteria are the most used systems for the expression of recombinant proteins, including MPs and the first hosts used prior to the other expression systems listed above, because they are easy to handle and inexpensive compared to eukaryotic systems. Furthermore, a wide range of genetic methods and vector systems are well-established. Among them, E. coli can be considered as the traditional and oldest bacterial gene expression system, which has been developed for many years and whose wide variety of plasmids and host strains are available. In most cases, the induction of gene expression is based Figure 1. The Nisin-Controlled Gene Expression (NICE) system in L. lactis. After the detection of nisin by the membrane-located sensor protein (NisK), this histidine protein kinase autophosphorylates and transfers its phosphate group to activate the cytoplasmic response regulator NisR . Activated NisR  subsequently induces transcription controlled by the PnisA promoter . After transduction,  and depending on the presence or absence of the corresponding targeting signals, the protein is either expressed into the cytoplasm or the membrane, or secreted into the external medium . B. chr: bacterial chromosome. Adapted from [12,25].
Well-characterized and highly versatile, the NICE system has been widely used for the over-expression and the subsequent functional and structural studies of homologous and heterologous MPs [12]. Moreover, it has been used for other purposes such as in pharmaceutical, medical, biotechnology, and food-technology applications [15,16,33]. Recently, the NICE system has been combined with the ZIREX system, allowing for the expression of different proteins at different times during the growth cycle [34].
Moreover, this NICE system has also been transferred to other Gram + bacteria (Leuconostoc lactis, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus plantarum, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus zooepidemicus, Enterococcus faecalis, and Bacillus subtilis) but without use for the high-scale production of MPs. Indeed, in many cases, regulated gene expression was established, but the growth of several species is retarded by the introduction of a special dual plasmid system, the different nisin sensitivity presented by the strains, the RNA polymerase sequence, and other factors influencing the expression of MPs [33]. After the detection of nisin by the membrane-located sensor protein (NisK) 1 , this histidine protein kinase autophosphorylates and transfers its phosphate group to activate the cytoplasmic response regulator NisR 2 . Activated NisR 3 subsequently induces transcription controlled by the PnisA promoter 4 . After transduction, 5 and depending on the presence or absence of the corresponding targeting signals, the protein is either expressed into the cytoplasm or the membrane, or secreted into the external medium 6 . B. chr: bacterial chromosome. Adapted from [12,25].
Well-characterized and highly versatile, the NICE system has been widely used for the over-expression and the subsequent functional and structural studies of homologous and heterologous MPs [12]. Moreover, it has been used for other purposes such as in pharmaceutical, medical, biotechnology, and food-technology applications [15,16,33]. Recently, the NICE system has been combined with the ZIREX system, allowing for the expression of different proteins at different times during the growth cycle [34].
Moreover, this NICE system has also been transferred to other Gram + bacteria (Leuconostoc lactis, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus plantarum, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus zooepidemicus, Enterococcus faecalis, and Bacillus subtilis) but without use for the high-scale production of MPs. Indeed, in many cases, regulated gene expression was established, but the growth of several species is retarded by the introduction of a special dual plasmid system, the different nisin sensitivity presented by the strains, the RNA polymerase sequence, and other factors influencing the expression of MPs [33].
In addition, the NICE system presents some drawbacks [20,33]: the maximum cell density obtained in a normal simple M17 acidifying buffered culture is about OD600 = 3 (1 g/L dry cell mass), lower than the density obtained from aerobic strains (100 g/L). The growth stops at a pH of around 5. Different techniques can be used to increase the cell density, including neutralization with NaOH or NH 4 OH, resulting in a maximum of OD600 = 15 (5 g/L dry cell mass), as well as growth under aerobic conditions in the presence of haem. Additionally, the proteolytic degradation of heterologous proteins is also a limiting factor in stable protein production; one recombinant strain has been constructed with the inactivation of the single protease HtrA, in which protein degradation is lower.

Host Strains Used for the NICE System
Different L. lactis host strains derived from L. lactis subsp. cremoris MG1363 can be used for the expression of cDNAs with the NICE system (Table 1, [28]). The most commonly used host strain for MP expression is the strain NZ9000. The nisin-producing strain NZ9700 [28] was obtained by the conjugation of the nisin-sucrose transposon Tn5276 of the nisin-A producer NIZO B8 with MG1464, a rifampicin-and streptomycin-resistant derivative of MG1363 [34]. Since the expression of MPs in L. lactis encounters difficulties due to low expression yields, different strategies have been developed to enhance their production. These strategies are either based on the introduction of an N-terminal fusion protein [19], mutations in the NisK ATPase domain of the sensor kinase (R406C) resulting in the DML1 strain [35], inactivation of the unique protease HtrA [36], selection of a strain enabling a higher plasmid stability (M4; [37]), or the overexpression of the cell envelope stress sensor/regulator CesSR [38]. Table 1. Bacterial strains and plasmids commonly used in the NICE system for the overexpression of MPs. nisA, nisRK, genes of the nisin operon; RifR, StrpR, and CmR: resistance to rifampicine, streptomycine, and chloramphenicol, respectively.

Characteristics References
Strains L. lactis
Mobitec Molecular Biotechnology DML1 NZ9000 strain transformed with pNZ-X-GFP-EmrC and selected by increased concentration of erythromycin. [35] Plasmids. The cDNA, or the gene encoding the MP of interest, is cloned into the appropriate expression plasmid, i.e., pNZ8048 or its derivatives (Table 1). These plasmids are based on the pSH71 replicon carrying the chloramphenicol resistance gene [31]. Plasmid pNZ8048 is the most commonly used plasmid for translational fusions. Genes of interest are directly fused to the NcoI site, which contains the ATG start codon directly downstream of the PnisA promoter. Different variants of pNZ8048 have been constructed: pNZ8148 is a shorter version of pNZ8048, with the deletion of a 60 bp heterologous DNA fragment from Bacillus subtilis, the initial cloning host of the pSH plasmid series [40]. pNZ8150 possesses a ScaI site directly upstream of the ATG start codon and therefore avoids the obligatory use of the NcoI site. Thus, it is no longer necessary to change the second amino acid of a protein if that codon does not conform with the sequence of the NcoI site. Other plasmids and strains are available and can be used for other purposes [20,33] (Mobitec Molecular Biotechnology; https://www.mobitec.com, accessed on 21 December 2021). The unidirectional cloning using classical restriction enzymes allows for a higher number of recombinant clones after transformation. Nevertheless, the MCS site is relatively small, containing less than 10 restriction sites, and partial digestions or mutagenesis is often required to obtain the desired constructs.

New Cloning Strategies
In addition to the classical cloning approaches, new strategies have been developed to overcome the problem of the low efficiency of gene manipulation in L. lactis and the instability of L. lactis-E. coli shuttle vectors [41,42]. Examples are ligation-independent cloning (LIC) and Gateway and other technologies developed by Berlec and collaborators. Furthermore, Geertsma and Poolman developed a generic cloning strategy compatible with high-throughput manipulations, which is also suitable for organisms other than L. lactis [43]. This method involves ligation-independent cloning (LIC) in an intermediary E. coli vector (pRExLIC-geneX), which can rapidly be converted via vector-backbone exchange (VBEx) into an organism-specific plasmid that is ready for high-efficiency transformation, such as pNZxLIC-geneX for L. lactis. In both LIC and VBEx procedures, rare restriction sites (SwaI and SfiI) were used. This strategy allowed for the successful expression of MPs from prokaryotic and eukaryotic origins [44][45][46].
Other laboratories developed strategies based on the Gateway technology (Invitrogen), which are now widely used to simplify the cloning of cDNAs into many different expression systems, from bacteria to eukaryotic systems [47], and for the high-throughput expression screening of integral MPs [48]. Several libraries are currently available in Gateway-compatible vectors [49]. However, L. lactis plasmids (e.g., pNZ8048 or derivatives) cannot be converted into Gateway destination vectors. Therefore, a strategy for the preservation of the correct reading frame has then been established for the rapid transfer of the cDNA from Gateway entry vectors into L. lactis nisin-inducible vectors [12,50]. This strategy allows for the successful expression of MPs from prokaryotic and eukaryotic origins, including proteins which could not be expressed using traditional cloning [7,51]. Only one development using an E. coli-L. lactis shuttle vector containing the Gateway cassette was proposed. These vectors allowed for the expression of two lactococcal phages, Tuc2009 and TP901-1 [52], and methyltransferases [53], but not of MPs.
Furthermore, in order to obtain a higher number of insert-containing plasmids after transformation, Berlec and Strukelj [54] developed a TA-cloning expression plasmid. A few years later, Berlec developed pNZ vectors for the dual expression of the proteins pNZDual and pNZDualTT, and one additional vector for the expression of proteins from polycistronic RNAs, pNZPolycist [55]. For the combinations tested, expression was higher using the latter compared to the pNZDual versions. Only one article showed the dual expression of secreted proteins fused to the usp45 secretion signal [56]. This point needs to be further investigated with different combinations of MPs to verify the impact of such constructs on the expression of MPs.
Once a gene is cloned within the proper vector, recombinant bacteria could be generated and used for MP expression through the NICE system.

Expression of Membrane Proteins Using the NICE System
In the last twenty years, the NICE system has proved to be highly versatile for the expression of proteins, including MPs using pNZ8048 and its derivatives. Up to now, 113 MPs from prokaryotic or eukaryotic origin, with diverse topologies and sizes, have been successfully expressed, including 79 in 2014 (12 and the present, Tables 2-4). This system also allows for the expression of MPs in their native oligomeric form (homo or heterodimers) [11,12].      Tables 2-4: List of homologous and heterologous prokaryotic and eukaryotic MPs expressed in L. lactis using the NICE system. Species, size, expression levels, and functions are given for each protein; the classification of MPs has been sorted according to the protein complexity in terms of numbers of TM helices. UNIPROT (http://www.uniprot.org/, accessed on 21 December 2021) was used as reference for protein information in addition to the literature.
Tables 2-4 display respectively non-exhaustive lists of prokaryotic MPs (homologous or heterologous expression) and eukaryotic MPs expressed in L. lactis with the NICE system. They include studies of functionally active proteins in which expression levels were not determined. The tables do not display, for some proteins, the percentage of expressed proteins when not available, and that of functional proteins out of the proteins expressed; indeed, this information is seldom reported since such a ratio is difficult to measure and necessitates isolating native proteins as controls. L. lactis MPs represent 20% of the total number of MPs expressed, while prokaryotic MPs are at 40%, and eukaryotic ones comprise 40%, respectively; among the latter, each origin (yeast, plant, and human) represents one-third ( Figure 2).
(Mus musculus); N. patriciarum (Neocallimastix patriciarum); S. cerevisiae (Saccharomyces cerevisiae); S. tuberosum (Solanum tuberosum). d The expression levels are given as a percentage of the recombinant protein compared to the total membrane proteins (TMP). Tables 2-4 display respectively non-exhaustive lists of prokaryotic MPs (homologous or heterologous expression) and eukaryotic MPs expressed in L. lactis with the NICE system. They include studies of functionally active proteins in which expression levels were not determined. The tables do not display, for some proteins, the percentage of expressed proteins when not available, and that of functional proteins out of the proteins expressed; indeed, this information is seldom reported since such a ratio is difficult to measure and necessitates isolating native proteins as controls. L. lactis MPs represent 20% of the total number of MPs expressed, while prokaryotic MPs are at 40%, and eukaryotic ones comprise 40%, respectively; among the latter, each origin (yeast, plant, and human) represents one-third ( Figure 2).  Tables 2-4 can be plotted according to the number of their TM helices and their molecular sizes. As shown in Figure 3, a large number of MPs have sizes below 100 kDa, with many MPs having either 6 or 12 TM helices, whether they are prokaryotic, from L. lactis or other bacteria, or are of eukaryotic origin (Figures 3 and  4), thus highlighting the two large families of proteins expressed (ABC and mitochondrial transporters).  Tables 2-4 can be plotted according to the number of their TM helices and their molecular sizes. As shown in Figure 3, a large number of MPs have sizes below 100 kDa, with many MPs having either 6 or 12 TM helices, whether they are prokaryotic, from L. lactis or other bacteria, or are of eukaryotic origin (Figures 3 and 4), thus highlighting the two large families of proteins expressed (ABC and mitochondrial transporters).

Expression of Prokaryotic MPs
Tables 2 and 3 report the successful expressions of 23 homologous and 43 heterologous MPs using the NICE system. The expression levels of prokaryotic MPs obtained were the highest of all reviewed MPs, with up to 30% of total MPs (TMP) by heterologous (HorA and MsbA) and homologous (LmrA) expression. The expressed MPs possess up to 13 TM helices, and even with such a high TM helix content, they were produced with expression levels of up to 20% TMP (BcaP and XylP). Most homologous MP expression studies have been focused on proteins belonging to the families of amino acid and ABC (ATP-Binding Cassette) transporters, probably related to the specialization of the laboratories working with this system. In addition to the above-mentioned amino acid and ABC transporters, other heterologous MPs have been expressed, belonging to diverse families such as cytochrome, permease, and binding proteins ( Table 3). The relatively high expression levels obtained with heterologous prokaryotic MPs could be explained by the fact that the codon usage is compatible with the AT-rich codon bias of L. lactis [107]. L. lactis also allowed for the expression of an MP with 14 TM domains, such as the MFS transporter called Rv1410 (Table 3; [85]).

Expression of Eukaryotic MPs
The expression of eukaryotic MPs in L. lactis was initiated and first reported in 2003 by Kunji and collaborators, together with the expression of mitochondrial carriers from yeast [11]. Since then, several other eukaryotic MPs from yeast, plants, and humans have been expressed, with levels from 0.1 to 10% of TMPs (Table 4), mainly from the mitochondrial carrier superfamily, but also from other families. Only one MP from protozoa (A. polyphaga; Table 4

Expression of Prokaryotic MPs
Tables 2 and 3 report the successful expressions of 23 homologous and 43 heterologous MPs using the NICE system. The expression levels of prokaryotic MPs obtained were the highest of all reviewed MPs, with up to 30% of total MPs (TMP) by heterologous (HorA and MsbA) and homologous (LmrA) expression. The expressed MPs possess up to 13 TM helices, and even with such a high TM helix content, they were produced with expression levels of up to 20% TMP (BcaP and XylP). Most homologous MP expression studies have been focused on proteins belonging to the families of amino acid and ABC (ATP-Binding Cassette) transporters, probably related to the specialization of the laboratories working with this system. In addition to the above-mentioned amino acid and ABC transporters, other heterologous MPs have been expressed, belonging to diverse families such as cytochrome, permease, and binding proteins ( Table 3). The relatively high expression levels obtained with heterologous prokaryotic MPs could be explained by the fact that the codon usage is compatible with the AT-rich codon bias of L. lactis [107]. L. lactis also allowed for the expression of an MP with 14 TM domains, such as the MFS transporter called Rv1410 (Table 3; [85]).

Expression of Eukaryotic MPs
The expression of eukaryotic MPs in L. lactis was initiated and first reported in 2003 by Kunji and collaborators, together with the expression of mitochondrial carriers from yeast [11]. Since then, several other eukaryotic MPs from yeast, plants, and humans have been expressed, with levels from 0.1 to 10% of TMPs (Table 4), mainly from the mitochondrial carrier superfamily, but also from other families. Only one MP from protozoa (A. polyphaga; Table 4; [100]) was expressed in L. lactis.

Membrane Proteins from Yeast (S. cerevisiae)
A total of 16 MPs from yeast have been successfully expressed in L. lactis. Two main studies on mitochondrial carriers revealed that all the MPs tested could be expressed with levels from 0.5 to 10% (Table 4). For some of them, expression levels were even improved by the rational design of the N-terminus (replacing or truncating these regions or by adding lactococcal signal peptides) [18].

Membrane Proteins from Plants
A total of 13 MPs from three plant species, i.e., A. thaliana, S. tuberosum, and N. patriciarum, have been successfully expressed in L. lactis. They belong to different families, for instance, an oxidase and various transport proteins (heavy metal, ATP/ADP, or sucrose), and their topologies span from peripheral to intrinsic 12 TM helices ( Table 4). The levels of expression obtained were relatively high, up to 30% (Table 4), without modifications of the sequence. These relatively high expression levels allowed for the performance of functional studies to discover and/or go deeper into the function of the MP expressed.

Comparison of Expression Levels between E. coli and L. lactis
The expression levels obtained for the expression of MPs in L. lactis are generally lower than those obtained for the overexpression of the same MPs in E. coli [7,12,60]. In some cases, the expression in L. lactis allowed for a higher expression or the expression of proteins usually produced in inclusion bodies in E. coli. For proteins produced with both bacterial expression systems, the levels were almost 10 times lower after expression in L. lactis as compared to that in E. coli [12]. The mechanisms for the production of proteins in both bacteria are different, and inclusion body formation allows E. coli to produce higher amounts of protein in inclusion bodies, mostly in-correctly folded or mis-folded, and therefore not functional. The slower protein synthesis is an advantage for L. lactis since it leads to the proper and correct functional folding acquisition of the MP produced in a single membrane. This slowness could be due to a limitation of amino acid import, especially for branched amino acids. This problem could be overcome by supplying the cells with an alternative path, such as a medium containing the appropriate dipeptides or by engineering the transport capacity of branched-chain amino acids [108]. Other strategies have been implemented using the optimization of functional expression, i.e., control of transcription rate, nutrient availability in richer medium, gene optimization, and/or fusion tags [60].
All MPs listed in Tables 2-4 have been expressed in L. lactis and were functional in this bacterium, which allowed for different assays to be performed and to decipher/discover the function of the MPs in the original organism.

Functional Expression of MPs
The following section will focus on examples of MPs of either prokaryotic or eukaryotic origin belonging to one functional class such as ABC transporters, secondary transporters, etc. L. lactis presents three major advantages over E. coli for functional MP expression: (i) it possesses only one membrane; (ii) it does not form inclusion bodies, and (iii) it expresses proteins in their native oligomeric state. Moreover, the genomes of MG1363 and NZ9000 are completely sequenced and annotated, allowing for the generation of mutated strains. These functional characterizations could be performed on: (i) whole bacteria using radioactive substrates, (ii) membrane vesicles, (iii) proteoliposomes after reconstitution with phospholipids, and/or (iv) solubilized/purified proteins. All MPs expressed (Tables 2-4) belong to different families: ABC transporters, secondary transporters, MPs originating from organelle (mitochondria, chloroplast), MCP, and other families ( Figure 5).

ABC Transporters
ABC transporters generally consist of four domains-two membrane-embedded domains carrying out substrate recognition and translocation and two hydrophilic nucleotide binding domains (NBDs). They represent one-third of the MPs expressed in L. lactis (Tables 2-4; Figure 5). Either transport or ATPase activities can be measured with radioactive or non-radioactive compounds on intact cells or detergent-purified protein within or outside of proteoliposomes or nanodiscs. In some cases, mutations allowed for the role assignment of certain amino acids to the proper function of the proteins. Studies in intact cells were facilitated by the availability of strains deleted in LmrACD, the three main ABC transporters present in the L. lactis membrane.

ABC Transporters
ABC transporters generally consist of four domains-two membrane-embedded domains carrying out substrate recognition and translocation and two hydrophilic nucleotide binding domains (NBDs). They represent one-third of the MPs expressed in L. lactis (Tables 2-4; Figure 5). Either transport or ATPase activities can be measured with radioactive or non-radioactive compounds on intact cells or detergent-purified protein within or outside of proteoliposomes or nanodiscs. In some cases, mutations allowed for the role assignment of certain amino acids to the proper function of the proteins. Studies in intact cells were facilitated by the availability of strains deleted in LmrACD, the three main ABC transporters present in the L. lactis membrane.
The ABC half-transporter LmrA (65 kDa, six TM helices), a well-characterized ABC transporter from L. lactis, was expressed in very high levels (up to 30% of TMP; [64]). The critical role of a carboxylate group in proton conduction to secondary-active transporters could be assigned [109]. Additional studies were performed on mutated versions expressed in L. lactis wild-type strains or strains with a deletion of LmrA homologs (LmrCD) [65]. Different studies based on nuclear magnetic resonance (NMR) and electron spin resonance (EPR) spectroscopy allowed for an understanding of the ATP hydrolysis cycle of the protein, the nucleotide binding, and the induction of the ion-motive force [110][111][112][113].
The thiamine high-affinity ABC transporter, ThiT (20 kDa, six TM helices), which belongs to the family of energy coupling factors, has been characterized in L. lactis. The expression levels in L. lactis was around 1-2% (Table 2; [45]). Mutagenesis studies allowed for the determination of some amino acids interacting with the energizing module, necessary for vitamin translocation [114]. EPR performed on purified ThiT and molecular dynamic studies allowed for a detailed description of the conformational changes of the protein during binding and coupling with the energizing module [83]. The structure of this protein was solved in 2014 [115]. The ABC half-transporter LmrA (65 kDa, six TM helices), a well-characterized ABC transporter from L. lactis, was expressed in very high levels (up to 30% of TMP; [64]). The critical role of a carboxylate group in proton conduction to secondary-active transporters could be assigned [109]. Additional studies were performed on mutated versions expressed in L. lactis wild-type strains or strains with a deletion of LmrA homologs (LmrCD) [65]. Different studies based on nuclear magnetic resonance (NMR) and electron spin resonance (EPR) spectroscopy allowed for an understanding of the ATP hydrolysis cycle of the protein, the nucleotide binding, and the induction of the ion-motive force [110][111][112][113].
The thiamine high-affinity ABC transporter, ThiT (20 kDa, six TM helices), which belongs to the family of energy coupling factors, has been characterized in L. lactis. The expression levels in L. lactis was around 1-2% (Table 2; [45]). Mutagenesis studies allowed for the determination of some amino acids interacting with the energizing module, necessary for vitamin translocation [114]. EPR performed on purified ThiT and molecular dynamic studies allowed for a detailed description of the conformational changes of the protein during binding and coupling with the energizing module [83]. The structure of this protein was solved in 2014 [115].
Moreover, out of the 31 ABC transporters that have been expressed in L. lactis, 19 originated from other bacteria. Among them, the half ABC-transporter MsbA from E. coli was expressed with a level slightly lower than that obtained from the homologous expression of LmrA (20-30%). This homodimeric transporter, with six TM helices and a molecular size of 64 kDa, is involved in lipid A export in E. coli [78]. Functional studies have demonstrated that substrate binding to the MsbA dimer caused NBD dimerization [116][117][118].
A heterodimeric ABC exporter, TM287/288 from Thermotoga maritima, has also been expressed in L. lactis [91]. TM287 and TM288, with a molecular size of 60 kDa and six TM helices each, form a functional heterodimer that shares 36% of its sequence identity with LmrCD, a well-characterized heterodimeric ABC exporter from L. lactis [65]. Functional studies determined that the NBDs only partially separate and remain in contact through an interface involving conserved motifs connecting the two ATP hydrolysis sites [91].
Finally, some eukaryotic ABC transporters were expressed in L. lactis. Among them, the well-known CFTR [42] and a plant mitochondrial ABC transporter, ATM3/ABCB25. Membrane vesicle assays revealed that glutathione (GSH) polysulfides are likely to be the substrates serving as precursors for iron-sulfur cluster assembly [92].

Secondary Transporters
Secondary active transporters exploit the electrochemical potential of solutes to shuttle specific substrate molecules across biological membranes, usually against their concentration gradient. These proteins are involved in the transport of amino acids [119], organic or inorganic anions, through symport or exchange processes [120]. MPs from the MFS superfamily were successfully expressed in L. lactis in their functional state [119,120]. Whilst the quantity of proteins produced in these studies was not determined, the biological activity of the proteins was detected using substrates specific to the transporters.

MPs from Organelle
A total of 26 MPs out of the 113 possess either chloroplast or mitochondrial origins (Tables 3 and 4, Figure 5). They belong to the families of ADP/ATP carriers (AAC) and Mitochondrial Pyruvate Carriers (MPC) in mitochondria and chloroplast, but also to other families in chloroplasts.

Mitochondrial MPs
AACs represent a large proportion of the MPs, with six TM domains expressed in L. lactis (Figures 3 and 4). Firstly, two mitochondrial carriers from S. cerevisiae, CTP1 and AAC3, have been successfully expressed at a level of 5% and shown to be functionally active in L. lactis [11]. Subsequently, ten other carriers from S. cerevisiae have been successfully expressed, with levels ranging from 1 to 10%, and activities varying depending on the substrate and the protein studied [18]. The relatively high expression levels obtained for these proteins could most probably be linked to the presence of cardiolipin in the membrane of L. lactis (32%; [103]). Indeed, it could be demonstrated that the expression of these proteins is facilitated, and the presence of the appropriate lipids could help to drive the protein folding to the right conformation.
The human isoforms of the ATP/ADP translocators (AAC1, 2, and 3) displaying number of TM helices and size features similar to the mitochondrial carriers of S. cerevisiae were also studied. AAC1, expressed at 0.5-1% of TMP, was sensitive to the same inhibitors as its yeast orthologs [103]. Mutants of this MP were shown to be involved in childhoodonset mild skeletal myopathy [104]. Zhang and collaborators [105] tested and compared the efficiency of L. lactis versus yeast mitochondria in order to study the impact of the inhibitors of AACs on the different isoforms. Their studies revealed that L. lactis shows a higher specificity in the exchange assay than yeast, that it allows for the differentiation between direct and indirect inhibitors, and that it is more reproducible and can be prepared in large quantities.
Among the mitochondrial proteins, the MCPs are remarkable. Indeed, the isoforms of MCP1 and MCP2 from three different species, i.e., the yeast Saccharomyces cerevisiae, Mus musculus, and Arabidopsis thaliana, have been expressed under a functional state in their heterodimeric form in L. lactis [96,97]. The mouse isoforms were able to transport pyruvate across the membrane in intact recombinant bacteria [97]. This uptake was sensitive to the mitochondrial pyruvate carrier inhibitor UK5099 and to 2-deoxyglucose, which collapses the proton electrochemical gradient. Moreover, artificially increasing the membrane potential by lowering the pH in the buffer from 7.2 to 6.2 significantly increased pyruvate uptake. The co-expression of mMPC1 and mMPC2 in the membrane of L. lactis was sufficient to allow for the import of pyruvate, with properties similar to the mitochondrial pyruvate carrier [121].

Chloroplast MPs
Expression in L. lactis using the NICE system proved to be efficient for the functional expression of several plant MPs involved in different chloroplast metabolic pathways, i.e., ceQORH, HMA6, and NTT1 proteins from Arabidopsis thaliana.
The peripheral ceQORH protein interacts with the chloroplast envelope through electrostatic interactions [122]. While this protein was produced in E. coli in inclusion bodies [122], it was expressed in L. lactis at almost 30% of TMP (Table 4; [50]), a surprisingly high expression level and similar to those obtained for homologous prokaryotic MPs (Tables 2-4). Functional characterization performed on purified proteins reconstituted in proteoliposomes revealed that ceQORH has NADPH-dependent dehydrogenase activity and requires a lipid environment. Moreover, when produced in L. lactis, ceQORH behaved as the natural chloroplast envelope protein and interacted with the bacterial membrane through electrostatic interactions [50].
Other chloroplast MPs such as the P1B-type ATPase family have also successfully been expressed, with levels from 0.7 to 3% of TMP (Table 4; [7,50]). These MPs (sixeight TM helices) translocate ions across plasma or organelle membranes at the expense of ATP consumption and are involved in the control of metal homeostasis within the cell [123]. Among the eight P1B-type ATPases encoded by the Arabidopsis genome, four have been successfully expressed in L. lactis [50]. Biochemical characterizations using phosphorylation assays were performed using L. lactis membranes expressing HMA6, and these assays allowed for the identification of this protein as a high-affinity Cu+ transporter of the chloroplast envelope [124].
The NTT1 protein is one of the AACs identified in the chloroplast; it imports ATP in exchange for ADP. This transporter has already been functionally characterized after expression in S. cerevisiae and E. coli [125,126]. Even though it was expressed at a very low level (0.2% of TMP), uptake assays of radioactive nucleotides could be performed on intact L. lactis cells and showed a time-dependent uptake of ATP, with a rate similar to the one measured in E. coli cells [50].
To conclude, L. lactis appears to be an appropriate expression system for the functional characterization of mitochondrial and Arabidopsis MPs, especially for chloroplast MPs. This can be explained by the fact that the L. lactis membrane contains cardiolipin and glycolipids [21], which are present in mitochondria [127] and the inner membrane of chloroplasts, respectively [128], in contrast to E. coli membranes [22], which have a different composition. The importance of the lipid composition of host cells in the overexpression of functional MPs has also already been underlined by other authors [3,23].

Membrane Proteins from Other Families
The first human MP produced in L. lactis was the KDEL receptor, Erd2. This protein with seven TM helices is involved in the retrieval of proteins of the endoplasmic reticulum (ER) at later stages of the secretory pathway. While expressed at a very low level, the protein could still bind its specific peptide and conserve the pH-dependent activities, as those in rat Golgi membranes [11].
Two MPs involved in human liver detoxification functions have been successfully expressed in L. lactis: the cytochrome-mono-oxygenase (CYP3A4) and the microsomal Glutathione S-Transferase 1 (MGST1). Interestingly, both proteins could be successfully expressed in L. lactis at higher levels than those previously obtained with classical expression systems (E. coli, S. cerevisiae) at 5 and 3% TMPs, respectively. This was also higher than results obtained for other eukaryotic membrane proteins expressed in L. lactis [51]. Expression of the MGST1 isoform from Rattus norvegicus in L. lactis was able to exhibit its GSH-transferase activity somewhat lower than values previously reported for rMGST1 from purified microsomes, or after heterologous expression in E. coli.
As discussed in the last two paragraphs, concerning the expression and functional characterization MPs in L. lactis, the number of MPs expressed in their functional state is increasing. Additional information has been obtained through the structural analysis of some of the proteins listed above.

Structures Resolved from MPs Expressed in L. lactis
Because of its numerous advantages in MP expression and functional characterization, L. lactis is now also a good alternative bacterial expression system for the structural determination of MPs of interest in E. coli. The first structure of a homologous MP expressed in L. lactis was obtained for OpuAC 10 years ago [129]. Then, the structure of ThiT was obtained, with both the wild-type and a selenomethionine-labeled protein. This crystal structure has been obtained at an expression level of 2% of TMPs [114,130]. One year after that, the same group resolved the structure of BioY, another L. lactis MP from the ECF family involved in biotin transport [59]. Altogether, almost 20 MP structures have been resolved in the last ten years after their expression in L. lactis using the NICE system, including their various conformations and bound to their substrates (Table 5). Most of the 3D structures were obtained by X-ray diffraction; only the last four ones for OpuA were obtained by single-particle cryo-EM (cryogenic electron microscopy), a new emerging technique that allows for the acquisition of structures without crystallization, reducing the required sample amount and allowing the usage of a wide arsenal of hydrophobic environments, a large advantage when working with MPs [136].
This opens up the road to the elucidation of other MP structures in the future since the expression levels obtained for almost all the proteins is close to 1-2% and higher (Tables 2-4). Furthermore, the ability to label the MPs with SelenoMet resolves the diffraction data [137] and the availability of specific protocols developed for this purpose [138].

Conclusions
Over the last two decades, Lactococcus lactis emerged and proved to be an alternative and promising expression system to other bacterial systems. Numerous prokaryotic and eukaryotic MPs with diverse topologies, origins, and functions were successfully expressed in L. lactis using the tightly regulated NICE system and at a level, although lower than E.coli, that still allowed for functional and structural characterizations. Finally, twenty crystal structures of MPs after expression in L. lactis were resolved and have thus opened up the road to others in the future. This promising cell factory will enrich the knowledge on MPs in their functional and structural states, and bring about the development of further biotechnological and biotherapeutical applications in the near future.